1. Stem Cells and the Art of Mesenchymal Maintenance

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Stem Cells and the Art of Mesenchymal Maintenance

Kevin C. Hicok and Marc H. Hedrick

these cells to determine similarity to hES cells generated via sexual reproduction has not yet advanced far. Furthermore, teratoma formation by hES cells remains a safety issue, so that large-scale clinical trials involving these cells cannot be undertaken until the safety issue is resolved.

Adults also have stem cells. Hematopoietic stem cells in the bone marrow that can recon- stitute the immune system have been known and studied for many years [13]. Stem cells in the liver allow rapid regeneration after liver surgery; stem cells in the dermis undergo con- tinuous cell division and differentiation to replace skin cells; and mesenchymal stem cells (MSCs) in bone provide osteoblasts for bone remodeling throughout life. Until the mid- 1980s, these stem cells were thought to be com- mitted to regenerating only the tissue in which they resided and were believed to be unable to differentiate toward cell fates not associated with their germinal layer of origin. Their potential as “true” stem cells was therefore not realized. In the 1990s, the molecular mechanisms involved in cellular differentiation began to be understood more fully. Moreover, development of in vitro differentiation assays helped cell biologists and tissue engineers realize the therapeutic potential of these cells. This chapter will review the successes, challenges, and future prospects of using stem cells in the tissue engineering of bone, cartilage, tendon, and ligament.

1.1 Introduction

The most promising emergent medical tech- nology of the early twenty-ﬁ rst century is stem-cell therapeutics. Traditionally, stem cells possess two important characteristics: the ability to undergo nearly unlimited self-renewal and the capability to differentiate into many (multipotent/pluripotent) or all (totipotent) mature cell phenotypes. The existence of stem cells and their ability to generate every tissue of the body during embryonic development has been known for many years. Transplant experiments performed in the 1970s, in which single stem cells were injected into early-stage blastulas, produced a chimera of donor and recipient cells in each organ of the resultant animal [29, 47].

The isolation and propagation of human embryonic stem cells (hES), however, has been achieved only relatively recently [111].

Political, moral, and ethical concerns surrounding procurement of these cells from embryos have held back their development as a source of cells for therapeutics or tissue engineering. Research efforts in the ﬁ eld of therapeutic cell cloning have skirted these issues by providing alternative methods, such as somatic cell nuclear transfer (SCNT), that generate hES cells without the use of intact embryos [46]. Until recently, the success rate of SCNT was extremely low, and the characterization of 1

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1.2 The Challenges of Mesenchymal Tissue Engineering

Unique challenges face those attempting to reconstruct or repair damaged bone, cartilage, ligament, or tendon. As the major support and connective tissues in the body, they must sustain high mechanical stress. All four tissues are largely devoid of cells and are made up mainly of extracellular matrix (ECM) proteins and minerals. Cartilage, tendon and ligament cells must all be able to survive in hypoxic conditions, because these tissues are largely avascular. As a result of this acellularity, these tissues, when damaged, often heal slowly, if at all. More- over, the body’s healing response diminishes with age [24, 28, 55, 73, 79, 84, 86, 100].

The tissues that orthopedic surgeons employ to repair damaged mesenchyme therefore have great demands on them. Success in using autologous or allogenic graft materials for mesenchymal tissue repair has been mixed, depending on the size and site of the wound or defect and the age and health of the patient. Autologous grafts for bone repair (the “gold standard” in orthopedics) have been more successful than autografts for cartilage, tendon, and ligament [15, 119]. For example, Brittberg and colleagues [15] reported positive results for 88% of patients in a clinical study of femoral condyle cartilage defect repair that used autologous chondrocyte-seeded grafts. On the other hand, the results for patellar transplants were less impres- sive, with only one third of the patients having a successful outcome [15].

Aside from the diffi culties associated with harvesting autograft material due to donor-site morbidity and the diffi culty of obtaining enough donor tissue, a major defi cit of autografts has been their frequent failure to become integrated with the surrounding host tissue.

Often the resultant chimeric tissue fails to attain the properties of the original tissue, so that secondary grafting procedures are needed.

Loading the graft material with mature phenotype cells has increased the amount of graft integration; however, limitations in the number of available autologous donor cells restrict the size of the graft that may be used [106].

Stem cells constitute an exciting alternative to the limitations of the current repair thera-

pies. Stem cells can undergo more than 50 rounds of replication and thus provide an abundant source of cells to repair or regenerate large regions of tissue. Because stem cells can differentiate into many different cell phenotypes, they can be used in situations where multiple tissues must be generated to restore organ function. In addition to providing a source of mature phenotypes, culture-expanded adult stem cells secrete paracrine factors that support vascularization of new tissue. Further- more, in instances where the cells themselves do not differentiate or produce a requisite factor for endogenous tissue healing or ex vivo regeneration, stem cells can be used to deliver gene therapy that in turn may enhance regeneration of the endogenous host tissue [12, 31, 92, 123]. These characteristics provide the mesenchymal tissue engineer with an abundant, renewable, and ﬂ exible source of cells that are capable of generating adequate amounts of ECM and of providing the enzymes, cytokines, and growth factors for the remodeling pro- cesses that are needed for the integration of implanted tissue.

1.3 Stem-Cell Repair of Bone

Recently stem cells of both embryonic and adult origins have been utilized. However, most early constructs utilized either endogenous or culture-expanded bone marrow-derived mesenchymal stem cells (BM-MSCs). In fact, orthopedic surgeons have, for many years, unknowingly utilized endogenous stem cells for bone repair. Early autograft transplant studies revealed the healing potential of bone marrow, soon recognized to contain a thera- peutically valuable mesenchymal cell population capable of generating osteoblasts [30, 88].

However, the identiﬁ cation and characterization of “stem” cells within this population has been accomplished only in the last 20 years [35, 90, 93].

When grown in vitro, these putative stem cells were found to reside principally within the adherent cell subpopulation. Researchers have taken advantage of this adhesive property to isolate and enrich the cells [16, 17, 30, 88, 90, 93]. This remains the principal way in which MSCs are enriched for use in tissue- engineering applications.

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To establish that stem cells are as multipotent, researchers have isolated single adherent cell clones that were then expanded in culture [41, 90, 93, 99]. Initial in vitro studies demonstrated that the BM-MSCs divide more than 50 times and differentiate into smooth muscle, osteoblastic, adipocytic, and chondrogenic phenotypes [90, 93] and, later, into tendon, ligament, and even cardiomyocytes (Fig. 1.1) [94, 124]. These cells can also be induced to display characteristics of endoderm and ectoderm tissues such as hepatic, neuro/

glial, endothelial, and epithelial markers [91, 97, 121]. Transplantation of labeled stem cells produced chimeric mice and demonstrated differentiation of the stem cells into cells of endodermal, ectodermal, and mesodermal origin [52].

The osteogenic potential of BM-MSCs has been extensively characterized in vitro [16, 17, 90, 93, 99]. Under appropriate conditions these

cells express genes from osteoblasts and synthesize proteins including type I collagen, bone sialoprotein, osteocalcin, osteopontin, and osteonectin. In vivo, BM-MSCs that are loaded either onto allogenic demineralized or mineralized bone matrix or onto to synthetic hydroxyapatite matrices generate osteoid tissue [19, 18, 60, 61, 63, 85]. It was therefore obvious that stem cells derived from human bone marrow would be a source of osteoblasts for bone tissue engineering.

In order for BM-MSCs to be used for generating tissue-engineered bone, two important problems had to be solved. First, BM-MSCs are quite rare; by some estimates there are as few as one to two cells per million isolated mono- nuclear cells [19, 93, 99]. It was therefore necessary to develop methods to increase the number of stem cells, while maintaining their osteo- potential. This was accomplished by Bruder and colleagues in 1996 [16, 17].

Figure 1.1. Adult adipose-derived stem cells (ADSCs) have the potential to differentiate into many different mature cell phenotypes. From top to bottom: Row 1: undifferentiated adult ADSCs in expansion culture, 10× phase objective. Row 2: ADSCs possess the ability to differentiate into adipocytes (left), osteoblasts (middle), and chondrocytes (right). Row 3: ADSCs also possess the ability to differentiate into neurons (left) and myocytes (right).

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Others searched for osteogenic stem cells in tissues such as skin, muscle, and fat [5, 6, 33, 50, 51, 104, 112, 125]. Of these, adipose tissue appeared to be the most promising, both eco- nomically and practically. Adipose tissue is abundant and relatively easy to harvest, and the number of stem cells that can be harvested from it is two to three log units higher per number of isolated cells than is the case for BM-MSCs.

When the number of these cells was increased, they differentiated into osteoblasts that synthe-

size bone matrix in vitro [37, 125] and in vivo [23, 38, 40, 92] (Fig. 1.2A). Even though adipose- derived stem cells (ADSCs) constituted an abundant alternative to BM-MSCs, it was necessary to do some ex vivo expansion prior to utilizing them in vivo [23, 38, 40, 92]. Recent data from our laboratories suggest that freshly isolated ADSCs may be loaded directly onto osteo- supportive matrices and can form bone in vivo (Fig. 1.2B). However, this treatment strategy still needs rigorous testing.

Figure 1.2. Bone formation by adipose-derived stem cells (ADSCs). Human ADSCs can generate new bone when implanted subcutaneously into immunodeficient mice (A). The cells were loaded onto hydroxyapatite scaffolds prior to implantation and were retrieved after 6 weeks. The scaffolds were processed and stained with hematoxylin and eosin. Noncultured ADSCs syn- thesized new bone (B with arrows) adjacent to the implanted scaffold (S) in a rat critical size defect (B). In the only case reported to date, primary ADSCs mixed with autograft were used to treat severe cranial defects in a nine-year-old female patient. Axial CT scans of her skull before surgery (C) and 3 months after surgery (D) reveal that significant mineralization has occurred in the defect site. Reproduced with permission from Lendeckel et al. [67].

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1.4 Microenvironmental Influences on Bone Formation by Stem Cells

Several factors are critical to the successful formation of bone by stem cells. The osteopoten- tial of a stem cell is signiﬁ cantly inﬂ uenced by environmental signals that include soluble growth and differentiation factors, as well as cell–cell and cell–ECM interactions. When stem cells are delivered either ectopically or into a critical size defect model in a soluble vehicle solution such as physiologically balanced saline, relatively few stem cells are actually found to multiply and differentiate into osteoblasts [60, 92]. One reason for this may be a lack of adequate environmental signals to direct the stem cells toward osteogenesis. However, when the stem cells are allowed to adhere to a bone or bonelike matrix, either alone or in the presence of endogenous signals such as bone morphoge- netic protein 2 (BMP-2), retinoic acid, dexamethasone, or 1,25-dihydroxyvitamin D3, bone formation is increased [23, 38, 92].

Various natural and artiﬁ cial scaffold materials have been utilized to serve as a delivery vehicle for stem cells and to provide the cells with appropriate cell–matrix interactions. Gen- erally, these scaffolds are composed of either autologous or allogenically derived bone, demineralized bone matrix, coral, collagen, calcium salts, or composites of these. Typically, the more similar a scaffold is to natural bone, the better it supports new bone growth. Thus, scaffolds containing tricalcium or bicalcium phosphate salts and hydroxyapatite appear to be most effective in supporting stem-cell osteogenesis. Studies performed in a canine segmental defect model illustrate how adult stem cells loaded onto an appropriate scaffold, a hydroxyapatite/β-tricalcium phosphate ceramic, can repair large gaps within long bones [18]. Other groups have utilized collagen-based scaffolds, poly(lactic acid) (PLA) polymers, and hydrogels, with variable success; however, these appear to be more suitable for cartilage formation [26, 38, 40, 61, 113].

Determining the optimal combination of soluble factor and matrix signals that gives rise to stem-cell osteogenesis is complicated. Stem- cell response to these signals may be model- and species-dependent. The length of time for

differentiation, the number of passages in culture, the health status of the stem-cell donor, and the tissue from which the cells have been obtained are all variables that inﬂ uence the ﬁ nal differentiation potential of the stem cells.

For example, culturing ADSCs on hydroxyapatite scaffolds in the presence of BMP-2 prior to implantation appears to aid in bone formation [23, 92], whereas in vitro stimulation of these same cells on the same scaffolds with other osteoinductive reagents, such as dexamethasone or 1,25-dihydroxyvitamin D3, may or may not aid in bone formation [38, 40]. Hattori and colleagues [52] demonstrated that human ADSCs are superior to undifferentiated cells for the formation of ectopic bone when seeded onto atelocollagen matrices cultured in the presence of dexamethasone, ascorbate, and β-glycerol phosphate. Hicok and colleagues, however, found that when dexamethasone and 1,25-dihydroxyvitamin D3 were used to pre- differentiate ADSCs, there was no beneﬁ t to ectopic bone formation [40].

The age of the donor from which the stem cells are derived may be important in determining the extent of predifferentiation required for effective bone formation. Mendes demonstrated that MSCs derived from either young or old donors, when implanted subcutaneously into nude mice, formed ectopic bone without dexamethasone pretreatment. However, dexamethasone signiﬁ cantly increased bone formation in implants that contained cells from individuals older than 50 years of age [78].

Other age- dependent factors, such as advanced glycation end products from elderly or diabetic recipients, inhibit stem cells from proliferating and differentiating into osteoblasts [62].

The concentration of osteoinductive factors and the length of exposure to them affect stem- cell effi cacy both in vitro and in vivo. ADSCs that were genetically modifi ed to express either constitutive BMP-2 or BMP-7 demonstrated increased levels of osteoid formation in comparison with stem cells cultured with these molecules [92, 123]. Epigenetic modifi cation of the stem cells may also be important, since com- pounds such as valproic acid, which has histone deacetylase inhibitory activity, have been shown to enhance osteogenesis of both adipose-derived and bone marrow-derived stem cells [22].

Species-speciﬁ c differences in the responsiveness of stem cells to their environment further complicate our understanding of which

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combinations of extracellular signals are most effective in inducing bone formation. Species differences in the response of osteoblast progenitor cells to osteogenic stimuli are known [21, 65, 69], but they need detailed characterization. Rat BM-MSCs readily adhere to and proliferate on an alginate gel surface, whereas human cells fail to adhere, unless type I collagen or β-tricalcium phosphate is added to the gel [64]. Srouji and colleagues [63] reported that BM-MSCs, when predifferentiated prior to transplantation in a rabbit tibia defect, gave rise to radiographically signiﬁ cant amounts of bone, whereas, as mentioned previously, human MSCs exposed to similar conditions did not substantially increase bone formation [108].

Stem cells are exposed not only to chemical stimuli and scaffold interactions, but also to physical forces that act on these cells during the engineering process and after transplantation. Limited studies have been performed;

however, application of physical force to the cells prior to transplantation seems to modulate their differentiation into osteoblasts. When human adult stem cells are exposed to either constant or intermittent mechanical or sheer stress, increased levels of osteogenic gene expression and mineralized matrix formation are observed [49, 59, 74, 77, 98].

Adequate oxygenation is critical to the successful generation and grafting of stem-cell- derived bone. Prior to implantation, cells must be adequately oxygenated so that they can expand into multiple layers and migrate into the inner surfaces of the delivery scaffolds.

Current culture systems cannot yet surpass the 150- to 200-µm limit of nutrient and oxygen penetration. For large defects in human long bones, for example, grafting tissues with thick- nesses in the millimeter range would signiﬁ - cantly decrease the time required for bone repair. To avoid cell necrosis, transplanted stem cell/scaffold constructs must be integrated rapidly into the recipient’s vascular system.

When growth factors such as vascular endothelial growth factor (VEGF), which stimulates vascularization, were made part of scaffolds, bone formation was found to be signiﬁ cantly enhanced [44, 66, 81].

Both adipose-derived and bone-marrow- derived stem cells can induce new blood vessel formation, because they synthesize physiologically signiﬁ cant amounts of angiogenic cytokines, including VEGF, placental growth factor

(PlGF), hepatocyte growth factor, and transforming growth factor β (TGF-β) [54, 57, 96].

Moreover, as in wound sites, these cytokines increase in quantity when the cells are exposed to hypoxic conditions [57, 58, 96]. When adult stem cells are placed into models of hind limb ischemia, collateral perfusion is increased [58].

Stem cells therefore not only can differentiate into osteoblasts, but may also support vascularization of the new bone. Understanding how this response is regulated is critical not only to engineering bone, but also to the successful utilization of stem cells in generating avascular mesenchymal tissues such as cartilage. It must be remembered that too high a level of oxygen within cartilage can induce apoptosis [72].

1.5 Safety and Success

An important challenge for the tissue engineer is to assess the safety of human stem cells when they are used to form bone in vivo. Even in severely immunocompromised rodent models such as the NOD/SCID mouse, there appears to be at least a low-level immunological response to the MSCs, to the scaffolds onto which they are seeded, or both [122]. This response depends on differences in how the cells are isolated or expanded in vitro prior to transplantation. It has been argued that the safety of autologous stem cells used for tissue engineering of bone in preclinical studies should be an adequate indicator of human stem-cell safety. Indeed, human BM-MSCs not only are immunoprivi- leged but also can suppress immune function [2]. In the end, however, the answer to this question lies in the results of clinical trials yet to be undertaken.

Notwithstanding the many as yet unan- swered questions, the use of human stem cells for bone repair has yielded encouraging initial results. Culture-enriched autologous BM-MSCs have been used to successfully treat refractory atrophic and hypotrophic nonunion fractures in a small phase I clinical trial in Spain [87].

Another case report from Germany describes how autologous ADSCs were used in combination with bone marrow to treat a nine-year-old girl who had sustained critical cranial defects as a result of trauma. Signiﬁ cant bone formation was demonstrated after only 3 months [67]. Previous attempts at using autologous and

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allograft bone alone had failed to heal these defects (Fig. 1.2C and D). As more clinical trials are conducted, bone derived from stem- cell grafts may make the challenges of autograft and allograft transplants a thing of the past.

1.6 Stem-Cell-Engineered Cartilage:

Microenvironmental Factors Influence Stem-Cell

Chondrogenesis

As with bone, embryonic and adult-derived stem cells can give rise to cartilage in vitro [27, 43, 56, 71, 93, 107, 116, 125, 126]. And similarly to bone, the ability of stem cells to form cartilage in vitro depends on both physical and chemical stimuli. These include growth and differentiation factors, cell–cell interactions, cell–matrix interactions, and inorganic chemical and physical factors such as oxygen tension and the three-dimensional organization of the cells. Unlike bone, however, the physical elements of the cartilage microenvironment appear to be more critical for stem cells to differentiate into chondrocytes than for stem cells to become osteoblasts.

Three-dimensional interactions between cells are required for chondrocyte differentiation and subsequent cartilage tissue formation.

When stem cells are plated as a monolayer, vir- tually no chondrogenesis results, even with added growth factors such as TGF-β and BMP [10, 27, 42, 45]. However, if the cells can establish three-dimensional polarity when cultured as condensed cell pellets or seeded into semi- solid matrices such as alginate or hydrogel, they express proteoglycans and collagen iso- forms, and a cartilage matrix is formed [11, 27, 71, 125]. The oxygen level in the culture is a second, important physicochemical parameter that affects chondrogenesis. Reducing the oxygen level in the culture to that which char- acterizes the cartilage environment in vivo enhances cartilage formation by ADSCs [14, 120], decreases cell proliferation, and increases the secretion of the essential protein, type II collagen, and of chondroitin-4-sulfate [120].

Stem-cell differentiation toward a chondrogenic phenotype also depends on activation of the TGF-β/BMP cell-signaling pathways [11, 71, 103, 126]. Thus, human ADSCs that had been predifferentiated in the presence of TGF-β in an alginate construct, when implanted subcutaneously, produced signiﬁ cantly more cartilaginous matrix than cells not so treated [27].

Other signaling mechanisms involving the parathyroid hormone-related peptide (PTHrP) receptor, glucocorticoid receptor, hyaluronic acid, and sonic hedgehog pathways have also been found to stimulate stem-cell chondrogenesis [25, 27, 32, 103]. The relative importance of growth and differentiation factors and of the resulting signaling pathways is, however, model-dependent. Therefore, the same devel- opmental challenges that must be overcome to generate bone also apply to stem-cell chondrogenesis [48].

Cartilage generation by stem cells also depends on the type of substrate or scaffold used. Conventional scaffolds are composed of collagen and proteoglycans or other hydrated organic molecules such as agarose, alginate, or hydrogels. Some articular cartilage defects, however, are repaired with the aid of constructs that contain calcium/phosphate salts [11, 32, 36, 80]. As shown in Fig. 1.3, a large articular cartilage defect in sheep was almost completely repaired when autologous BM-MSCs were loaded onto a β-tricalcium phosphate scaffold [36].

Understanding the properties of the scaffolds or matrices in which the cells are delivered is especially critical for cartilage formation in vitro and its implantation. The microenvironment within the scaffold must be such as to support stem-cell growth and differentiation.

As discussed in other chapters of this volume, the scaffold material itself must have physical properties comparable to those of the host cartilage and last until enough new cartilage has been produced to replace or supplement the implanted scaffold. For the scaffold to be replaced, it must be biodegradable.

Gelatin and agarose-based scaffolds have been used most commonly. When stem cells are seeded into scaffolds with nonosteogenic matrices such as gelatin and agarose, the matrices undergo changes in stress and compression that correlate with increased cartilage matrix accumulation. [11] When ADSCs are loaded onto gelatin scaffolds, their equilibrium

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compression and shear moduli increase after 28 days of culture, but this does not occur when they are loaded onto hydrogel-based matrices [11].

The efﬁ cacy of stem-cell chondrogenesis is model-dependent. Direct addition of stem cells to articular cartilage defects in rabbit and dog condyles generated tissue that was histologi- cally comparable to native tissue [1]. However, both bone-marrow-derived and adipose- derived cartilage displayed less strength and elasticity than native cartilage. Direct addition of BM-MSCs in a caprine osteoarthritis model reduced cartilage loss and induced cartilage regeneration [80]. Cells derived from autologous rabbit bone marrow were able to regenerate a femoral condyle defect when loaded in a collagen gel [117]. Twenty-four weeks after transplantation, the reparative tissue from the BM-MSCs was stiffer and less compliant than the tissue derived from the empty defects, but it was less stiff and more compliant than normal cartilage [117]. Poly(lactic-co-glycolic acid)

(PLGA) scaffolds supported cartilage formation by BM-MSCs transplanted into rabbit patellar defects [114]. Stem cells derived from allogenic rabbit adipose tissue, when delivered in a ﬁ brin matrix, formed cartilage in an articular condyle defect that, on histological examina- tion, appeared to have become integrated with the surrounding host tissue [82]. However, the new tissue became degraded after 12 weeks.

Gao and colleagues [32] recapitulated the host microenvironment and had greater long- term success. They utilized a two-layered matrix composed of a bottom layer of inject- able calcium phosphate and a top layer of hyaluronan and found that by 12 weeks the defects had become ﬁ lled with a stratiﬁ ed osteochondral tissue that was integrated into the surrounding tissue.

Alhadlaq and colleagues created a composite human articular condyle by predifferentiating BM-MSCs along the chondrogenic or osteogenic pathways and then loading the cells into photopolymerization gels [3]. The mold was Figure 1.3. Bone marrow-derived mesenchymal stem cells (BM-MSCs) form new articular cartilage in vivo. At 12 weeks postoperation, the defects in the BM-MSC group were mostly repaired with tissue-engineered cartilage, resulting in a relatively smooth and consistent joint surface (A). At 24 weeks postoperation, the regenerated area was covered by smooth, consistent hyaline tissue that was indistinguishable from the surrounding normal cartilage (B). The defects in control group 1 were partially repaired with fibrous tissue, leaving some depression in the defect areas (C). In control group 2, a thin layer of red, irregular tissue surfacing the defects can be seen, and cracks on the surrounding normal cartilage are obvious (D). Reprinted from Guo et al. [36]. Copyright 2004, with permission from the European Association for Cranio-Maxillofacial Surgery.

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made from a human condyle, photopolymer- ized, and then transplanted subcutaneously into immunocompromised mice. After 4 weeks, the resultant construct retained both the shape and the dimensions of the condyle and contained osteoid and cartilaginous matrix. More complex scaffolds can take advantage of stem- cell multipotentiality and may better stimulate the host environment, thereby providing appropriate niches for both bone and cartilage repair.

Spinal disc repair represents another fi eld for the use of stem cells in cartilage tissue engineering. Cultures of ADSCs that also contain nucleus pulposus cells give rise to type II collagen and to aggrecan that is typical of nucleus pulposus cells [68]. In conventional spheroid cultures, adult MSCs express genes typical of intervertebral disc nucleus pulposus cells, including type II collagen, aggrecan, decorin, fi bromodulin, and cartilage oligomeric matrix protein, with the levels of expression typical of disc cells rather than of hyaline articular cartilage [109]. In contrast, chondrogenically induced stem cells express type X collagen, an indicator of chondrocyte hypertrophy and eventual ossifi cation [83]. Ossifi cation in articular cartilage repair is necessary for tissue integration with the surrounding bone tissue, but in disc regeneration, ossifi cation of the tissue is undesirable. However, if the surface properties of the substrates on which the stem cells are grown are altered, type X collagen gene expression in BM-MSCs can be inhibited [83]. Whether this also induces the expression of the desirable proteoglycan proteins remains to be determined.

A number of other stem-cell-dependent variables are likely to infl uence the effi cacy of the stem cell/scaffold constructs, and studies to identify these variables are therefore war- ranted. For example, the site from which stem cells are harvested may infl uence their chondrogenic potential. Adult stem cells derived from bone marrow and those derived from adipose tissue appear to differ in their ability to form cartilage in vitro [42, 48]. The reasons for these differences and whether the observed differences are relevant in vivo remain to be determined but may be important in planning future therapies.

As yet, the mechanical properties of stem- cell-derived cartilage have not been characterized for most model systems. Tissue strength

and viscoelastic, tribological, and anisotropic properties must be assessed to determine whether the new tissue can withstand in vivo stress loads. Secondly, appropriate studies are needed to establish the number of cells needed per scaffold for the formation of adequate amounts of cartilage, while avoiding necrosis or apoptosis. If too many cells are implanted into a wound, tear, or defect, the implant will not be sustained because of insufﬁ cient amounts of nutrients in the surrounding avascular, acellular matrix.

Stem cells from different body sites should be evaluated systematically for their chondrogenic potential [48]. The evaluation should take into consideration the relative ease of obtaining the stem cells, donor site morbidity, and the requirements for ex vivo expansion, as well as the quantitative and qualitative differences in the efﬁ cacy of the engineered tissue generated by the cells. Recent attempts to address these issues include reports [102, 103] that stem cells derived from synovium produced more cartilage than BM-MSCs, periosteal progenitors, skeletal muscle, and ADSCs from the same donor.

The therapeutic potential of cartilage syn- thesized by stem cells is illustrated by a report of two cases in which human BM-MSCs were successfully used to treat patellar articular cartilage defects, with the two individuals report- ing that they had less joint pain after 1 and 2 years of follow-up [118]. Arthroscopy of the injured sites showed that they contained ﬁ bro- cartilage [118].

1.7 Keeping Things Together:

Stem-Cell-Engineered Ligament and Tendon

The use of stem cells to generate tissue-engineered ligament and tendon holds great promise. The cost of ligament repair alone exceeded ﬁ ve billion dollars in 2002 [89]. The current “gold standard” for repairing the most commonly injured ligament, the anterior cruciate ligament, is by implantation of autografts that consist of either patellar tendon or two hamstring tendons that are harvested at the time of surgery [115]. The rates of failure and recurrence of anterior cruciate ligament injury

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treated by these autograft methods, however, are still unacceptably high. As is the case for chondrocyte-loaded cartilage autografts, the supply of autologous tenocytes and ligament ﬁ broblasts is limited, and their harvest often leads to donor-site morbidity.

The ﬁ eld of stem-cell-engineered tendons and ligaments is still in its infancy, even though the observation that BM-MSCs can differentiate into tendons and ligaments was made over a decade ago [20]. As is the case for cartilage and bone, the abundance of stem cells makes up for the limited availability of donor tissue and the high donor-site morbidity. Stem-cell- generated grafts, however, like ligament ﬁ broblast- and tenocyte-seeded grafts, must be able to synthesize and remodel collagen, elastin, and other ECM proteins so that physiologically relevant levels of mechanical resistance and organization can be attained. Secondly, they must be delivered on a scaffold that is initially strong enough to endure cyclic stresses yet can undergo gradual degradation, thereby allowing the stem cells to differentiate and to secrete matrix proteins that can replace the scaffold.

Finally, the new tissue must integrate with the host tissue so as to avoid recurrence of the injury.

Identifying optimal in vitro conditions that permit implantation has been challenging.

Three factors seem essential: the absolute number of stem cells, the ratio of cells to collagen, and the ability of the cultured cells to synthesize the collagen in vitro prior to implantation [8, 9, 53]. Furthermore, as with ligament ﬁ broblast-loaded constructs, exposure to appropriate cyclic strain is important to establish appropriate orientation and cross linking of matrix ﬁ bers within the new tissue [34].

To date, the characteristics that have been attained by stem-cell-engineered tendons and ligaments have fallen short of the desired outcome. In early studies, BM-MSCs initially seeded onto collagen scaffolds did not induce ligament regeneration, because the collagen scaffold did not stimulate the stem cells to produce adequate amounts of ligament matrix.

In addition, the collagen ﬁ ber scaffolds did not support long-term anchoring of the grafts in vivo [115]. In a rabbit full-length, full-thickness tendon-defect model, the average maximum force and stress values of the BM-MSC- engineered collagen implants were approxi- mately 30% that of normal patellar tendons

[53]. The average repair stiffness and modulus values were 30% and 20%, respectively, of the values in normal patellar tendon [53]. Simi- larly, rabbit BM-MSCs loaded onto collagen gels and contracted onto sutures possessed only 25% of the maximum stress capacity of the normal tendon when implanted in a patellar tendon defect model. More disconcerting was the observation that bone formed in 28% of the patellar implant sites [7]. The results point to the obvious conclusion that the mesenchymal tissue engineer must continue efforts to identify the relevant mechanisms involved in tendon and ligament differentiation.

The recent utilization of silk-fi ber-based delivery scaffolds with BM-MSCs has improved stem-cell-engineered ligaments [4, 115]. The silk fi bers have superior mechanical properties and biodegrade within a more compatible timeframe. When woven into a six-cord rope confi guration, the constructs display mechanical properties similar to the anterior cruciate ligament, and the constructs possess a greater surface area for cell attachment and ECM depo- sition [4, 115].

Integration of tendons and ligaments into the bone is critical for the long-term success of any engineered graft. Autografts and allografts used for ligament and tendon recon- struction have a poor record in this regard.

Because of their ability to differentiate into multiple tissues, stem cells have the potential to generate the different tissues required for appropriate integration into the host tissue.

When tendon autografts coated with fi brin glue were loaded with MSCs, cartilage cells covered a large area at the tendon–bone junc- tion within 2 weeks [70]. By 8 weeks, a mature zone of cartilage blended from bone into the tendon grafts. At 8 weeks, the MSC-enhanced grafts had a signifi cantly higher failure load and greater stiffness than the grafts loaded with fi brin glue.

For stem-cell-based therapeutics to be a success in clinical trials, research must be done to address key factors known to infl uence stem- cell effi cacy. For example, autologous stem cells may be infl uenced by both the health status and the age of the patient. Bruder and colleagues have observed that the number of stem cells in bone marrow appears to decline with age [17];

however, whether this is true of adult stem cells from other body sites remains to be determined. Fewer stem cells are available as an

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individual ages, but their ability to proliferate remains the same [39, 110]. Therefore, for repairs in the elderly, more stem cells have to be harvested; alternatively, allogenic cells can be used.

Although the effects of aging on the ability of human stem cells to form tendon and ligament are unknown, an intradonor rabbit study utilizing BM-MSCs extracted from animals 1 and 4 years of age found no statistically signiﬁ cant differences in the mechanical properties of tendon regenerated by cells from the younger and the older animals. The stem cells from the older animals, however, exhib- ited reduced mechanical properties. Therefore, banking stem cells early in life for later use may lead to a better outcome. When the clinical and biomechanical factors involved in tendon and ligament differentiation are understood, tendons and ligaments grown from adipose or bone marrow cells are likely to become commonplace.

1.8 The Answers Are on the Horizon

Bone, cartilage, tendon, and ligament engineered from stem cells hold great promise to reduce suffering resulting from orthopedic injury and disease. With proper selection of stem cells and an appropriate supply of environmental signals, outcomes approaching 100% recovery may become possible. Indeed, mesenchymal tissue-engineered therapies that use novel combinations of scaffolds, stem cells, and differentiation factors are being reported almost monthly.

These novel approaches represent attempts to overcome the limitations of conventional stem-cell delivery systems. For example, replac- ing collagen with silk ﬁ bers generates porous silk ﬁ broin scaffolds that are biodegradable and stronger than collagen scaffolds and that can support higher rates of human stem-cell differentiation than can conventional scaffolds [75, 76]. When BM-MSCs were loaded onto a biodegradable scaffold embedded with DNA that encodes an osteodifferentiation factor (BMP-4) and a proangiogenic factor (VEGF), greater amounts of properly vascularized bone were formed than when scaffolds containing

only stem cells or one factor alone were used [44].

An exciting use of adult stem cells in mesenchymal tissue engineering is to take advantage of subtle differences found among cells from different body sites [95, 101]. Shi and colleagues recently described the isolation, characterization, and propagation of stem cells from different regions of adult human dental tissues that, when combined with appropriate scaffolds, developed into tissues resembling bone, dentin pulp, and cementum [105]. From an industry perspective, multiorigin stem-cell- engineered tissue poses signiﬁ cant intellectual property and regulatory hurdles for those who are brave enough to attempt to bring such tissue to the medical community. Ultimately, however, this approach may provide the regen- erative capacity needed fully to restore or replace a damaged organ.

These considerations lead to what is perhaps an obvious conclusion: as tissue engineers identify and implement the essential multi- factorial requirements for growing new or ﬁ xing old mesenchymal tissues, the full therapeutic potential of stem cells may ultimately be realized.

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